WO1997049718A2

WO1997049718A2 - Conjugates of an oligonucleotide/electronic conductor polymer with a molecule of interest, and their uses

Info

Publication number: WO1997049718A2
Application number: PCT/FR1997/001134
Authority: WO
Inventors: Joseph Marchand; Hervé Bazin
Original assignee: Cis Bio International
Priority date: 1996-06-25
Filing date: 1997-06-25
Publication date: 1997-12-31
Also published as: JP4803851B2; FR2750136A1; JP2000514786A; EP0912593B1; US6160103A; EP0912593A2; KR20000022148A; FR2750136B1; DE69706989D1; ATE206132T1; CA2258802A1; WO1997049718A3; DE69706989T2; CA2258802C; AU3448497A

Abstract

The invention concerns a macromolecule of formula (I): P-O-M in which M represents a molecule of interest; O represents an oligonucleotide chain; P represents a monomer of an electronic conductor polymer, and x and y are integers equal to 1 or more. The invention also concerns the copolymers obtained from the P-O-M macromolecules.

Description

CONJUGATES OF AN ELECTRONIC CONDUCTIVE OLIGONUCLEOTIDE / POLYMER WITH A MOLECULE OF INTEREST, AND USES THEREOF.

The present invention relates to new methods and new compounds for controlling the binding of different molecules of interest to an electronic conductive polymer (PCE);

PCT application WO 94/22889 in the name of CIS BIO INTERNATIONAL (Inventors TEOULE et al.), Describes the fixation of oligonucleotides on a support consisting of an electronic conductive polymer (PCE). This fixation can be done in different ways:

1) By fixing a presynthesized oligonucleotide on the PCE (either by chemical reaction of 1 Oligonucleotide on the PCE previously functionalized, or by copolymerization of the PCE monomers with the condensation product of 1 Oligonucleotide on one of said monomers). 2) By elongation of 1 oligonucleotide from a nucleoside, a nucleotide, or an oligonucleotide already attached to PCE, using for example one of the conventional methods of synthesis of nucleic acids.

The attachment of oligonucleotides to PCEs makes it possible to facilitate the obtaining and the use of oligonucleotide matrices, which are in particular usable for nucleic acid sequencing and diagnosis.

Like the oligonucleotide matrices, the peptide matrices, and more generally, matrices of various molecules indeed constitute a particularly interesting tool, for example in the field of diagnosis, or for the screening of active molecules. It would therefore be desirable for other types of matrices to benefit from the improvements brought about by the use of PCE.

However, the attachment to a PCE of molecules of interest other than the oligonucleotides, for example the attachment of peptides, poses more problems than the attachment of oligonucleotides. Indeed, although methods for the synthesis of pyrrole carrying an amino acid or a dipeptide are described in the literature [GARNIER et al. J. Am. Chem. Soc, 116, 8813-8814 (1994)], these are synthesis methods in solution which, in practice, do not make it possible to obtain, with a sufficient yield, synthetic peptide molecules larger than 2 or 3 amino acids. However, the molecules presenting a real interest in the field of diagnosis or that of screening active molecules are longer; for example, a "minimal" antigenic motif usually has an average of 6 amino acids.

The usual methods of peptide synthesis on solid support, derived from the MERRIFIELD technique, involve different groups protecting the side chains of amino acids. The cleavage of these groups, as well as the separation of the peptide and the support at the end of the synthesis are carried out in a strong acid medium (hydrofluoric acid or trifluoroacetic acid). However, in an acid medium, the PCE monomers, and in particular the pyrrole residue, tend to polymerize, thus creating undesirable by-products. It therefore appears that neither the synthesis method in solution, the possibilities of which are limited to the synthesis of di- or tripeptides, nor the synthesis on a support, the implementation of which requires chemical conditions incompatible with the stability of the pyrrole residue, are not suitable for the synthesis of peptides modified by a pyrrole residue. It is however possible to graft a PCE monomer, for example a pyrrole residue, onto a preformed oligopeptide derived from a traditional peptide synthesis on solid phase. This grafting can be done by known methods, by reaction between a carboxylic derivative of pyrrole (activated by a coupling reagent) and one of the available amino functions of a peptide (for example the amino function of the N-terminal end) following the diagram below [SE WOLOWACZ et al., Anal. Chem., 64, 1541-1545, (1992)].

Pyrrole— COOR + NH2— peptide> Pyrrole— CONH— peptide

At the end of the reaction, the pyrrole-peptide conjugate must be isolated from the reaction mixture containing the unreacted peptide and pyrrole, as well as any salts and by-products.

The different methods which can a priori be considered for this purpose are those usually used for the purification of peptides, and in particular the reverse phase chromatography (RP-HPLC) or gel filtration methods. The simplest way to detect peptides after chromatography is to measure the absorption in the ultraviolet, at a wavelength of 215 nm to 220 nm; indeed, at this wavelength all peptides absorb light. However, this measurement of the absorption around 215 nm has the drawback of being insensitive, and of not being specific, since solvents as well as organic or inorganic ions are also detected. There are methods for specifically detecting peptides, by a chemical reaction "in line" at the outlet of the chromatography column. This method has the advantage of increasing the sensitivity of the detection, but increases the implementation, and furthermore irreversibly modifies the peptide; this modification can in particular have important consequences on these functional properties (for example its antigenicity).

The problems set out above in the case of peptides also arise for the detection of other molecules of interest, etc. Indeed, many substances, such as sugars, polysaccharides, steroids, or else do not have specific absorption at a wavelength determined in UV, or have only a weak absorption, which affects the sensitivity of the detection.

We are then led to detect, by an appropriate physical or chemical means, the presence of the molecule sought in each fraction resulting from the chromatography, which obviously takes a long time; moreover, this analysis is often destructive of the analyte concerned.

The present invention aims to obtain conjugates, easy to synthesize and purify, of molecules of interest with a PCE monomer. To this end, the inventors have prepared conjugates having properties which neither the molecule of interest nor the PCE monomer naturally possess. In these conjugates, the molecule of interest and the PCE monomer are linked via an oligonucleotide chain, acting as a spacer arm between the PCE monomer and the molecule of interest considered.

The subject of the present invention is a macromolecule of formula (I) below: P-O-M (I) in which:

M represents a molecule of interest; O represents an oligonucleotide chain; P represents a monomer of an electronic conductive polymer.

For the purposes of the present invention, the term “molecule of interest” means any molecule having a functionality useful in reactions on solid support, for example synthesis reactions, or direct or indirect detection. This molecule of interest can be, for example, without this list being limiting, a biomolecule, such as a protein (in particular an enzyme) an amino acid, a peptide, a glycopeptide, a lipid, a steroid, a glycolipid , a sugar, a polysaccharide, a molecule capable of generating, directly or indirectly, a signal, or else a complex, multifunctional molecule; etc. Advantageously, this molecule of interest constitutes one of the members of an affinity couple, it may for example be biotin, or a potentially antigenic peptide, etc. P can for example be a monomer of polyacetylene, polyazine, poly (p-phenylene), poly (p-phenylene vinylene), polypyrene, polypyrrole, polythiophene, polyfuran, polyselenophene, polypyridazine, polycarbazole , polyaniline, etc.

Advantageously, P is a pyrrole group. The oligonucleotide chain O can consist of the assembly of natural nucleotides and / or of nucleotide analogs, such as those described for example by UHLMANN, [Chemical Review, 90: 4, 543-584 (1990)]. It may be a single-stranded oligonucleotide, or a double-stranded oligonucleotide over at least part of its length. In the second case, one of the strands is covalently attached to the monomer P, and the other strand is covalently attached to the molecule of interest M.

Theoretically there is no limitation in the nature or the length of the oligonucleotide; in practice, the oligonucleotide chain will advantageously have a length of between 6 and 60, preferably between 10 and 30 nucleotides. Advantageously, the percentage in (G + C) of the oligonucleotide O is less than or equal to 70%, and preferably less than or equal to 50%.

The binding of the oligonucleotide O to the PCE monomer can be carried out as described in PCT application WO 94/22889. The attachment of the molecule of interest M to the oligonucleotide O can be carried out by various methods, known in themselves, making it possible to link an oligonucleotide to another molecule. The choice of the most suitable method essentially depends on the nature of the molecule of interest M.

For example, the oligonucleotide O can be activated by attachment of an amino acid N-hydroxy succinimide ester of an SH group [ARAR et al., Bioconjugate Chem. 6, p. 573-577, (1995)], or a maleimide group.

The present invention also relates to a process for preparing a POM macromolecule as defined above, from a mixture comprising said macromolecule as well as the P-0 and M reagents from which it was formed, which process is characterized in that it comprises at least one step during which one proceeds to the fractionation of said mixture, by any means allowing to separate the fractions comprising respectively M and PO M, of the fraction comprising PO, and a step during which the fraction comprising POM is detected, and / or the quantity of POM is determined by detection and / or measurement of an associated parameter with the oligonucleotide O and not associated with the molecule of interest M. The oligonucleotide chain O plays, by its chemical nature and by its length, a role in the physical properties (polarity in chromatography, for example) of the conjugate with the molecule of interest considered, which makes it possible to adjust an optimal separation of the conjugate obtained and the excess reagents. On the other hand, it allows the specific detection of the conjugate in the ultraviolet, at a wavelength between 240 and 270 nm (advantageously, 265 nm), and optionally by hybridization with an oligonucleotide of complementary sequence.

The properties conferred by the oligonucleotide chain O thus allow easy manipulation of minute quantities of molecules of interest, by facilitating their separation by methods of ultrafiltration, and / or partition chromatography, or affinity chromatography on oligonucleotides of complementary sequence, as well as their detection and rapid quantification by a specific absorption measurement between 240 and 270 nm. This is particularly advantageous when the molecule of interest M is a peptide; in fact, the measurement of the oligonucleotide absorption at 265 nm is much more sensitive and specific (it is not disturbed by salts and solvents) than that of the peptide absorption at 205 nm.

The present invention also relates to copolymers of which at least one of the units consists of a P-O-M macromolecule as defined above.

Polymers in accordance with the invention are, for example, defined by one of the following formulas (II), (III) or (IV):

M

^?

^? (IV)

M in which: x and y represent whole numbers equal to or greater than 1, and M represents a molecule of interest; O represents an oligonucleotide chain

P represents a monomer of an electronic conductive polymer.

In a copolymer in accordance with the invention, the monomers P of electronic conductive polymer can be identical to each other, or else of a different nature; the oligonucleotides O can also be identical to each other, or different in their sequence and / or in their size, and / or their single- or double-stranded nature; in the same way, the molecules M can be identical or different between them.

These copolymers can be prepared by copolymerization of one or more purified POM conjugate (s), or as defined above, with P monomers, and / or with PO conjugates, such as those described in PCT application WO 94/22889, and / or with PM conjugates; they can also be obtained by fixing one or more molecules of interest M to all or part of the lateral oligonucleotide chains of a PCE / oligonucleotide copolymer, such as those described in PCT application WO 94/22889. This fixation can for example be carried out by covalent bonding of a molecule of interest M with an oligonucleotide O, itself attached to PCE, or else via an oligonucleotide hybrid with said oligonucleotide O on at least part of its length. The copolymers in accordance with the invention can be used in all the applications in which it is usual to fix molecules of interest on a solid support, and in particular on an electrode.

The present invention relates to electrodes, the surface of which carries at least one copolymer in accordance with the invention.

For example, the surface of an electrode according to the invention can be entirely covered with a single copolymer according to the invention; it can also carry several copolymers in accordance with the invention, and differing from one another by at least one of the constituents P, O, or M,; this or these copolymers can also be combined, on the same electrode, with other polymers, for example PCE / oligonucleotide copolymers, such as those described in PCT application WO 94/22889, or else with polymers of different nature, such as for example PCEs resulting from the polymerization of P monomers as defined above. "

Polymers in accordance with the invention can be used for the constitution of arrays of molecules of interest, in particular of arrays of electrodes comprising at least one electrode according to the invention, as defined above.

Electrodes constituting different points of the same matrix can differ from each other by the monomers P entering into the composition of the copolymers present on their surface, and / or by the oligonucleotides O and / or the molecules M of the side chains of these copolymers; they can also differ from each other by the quantity of these side chains per unit of area.

Some of the electrodes of the same matrix can carry a polymer other than a copolymer in accordance with the invention, for example, a PCE / oligonucleotide copolymer such as those described in PCT application WO 94/22889. Depending on the use envisaged, this PCE / oligonucleotide copolymer can be stored as it is, or else be used for fixing other molecules of interest, directly, or by hybridization with another oligonucleotide carrying the molecule of interest.

Such matrices can be prepared by proceeding with the targeted deposition of the desired polymers on specific electrodes; for example, copolymers in accordance with the invention can be deposited by electrochemical copolymerization addressed, on the chosen electrodes, of POM conjugates, and / or of variable amounts of the same POM conjugate, with the monomers P, and optionally PO conjugates , and PM. The use of POM macromolecules and copolymers in accordance with the invention therefore makes it possible to easily obtain multifunctional matrices. The POM conjugates according to the invention can also be used to calibrate matrices of oligonucleotides fixed on a support of electronic conductive polymer. The inventors have in fact found that a reproducible correlation can be established between the quantity of O-M side chains, and the quantity of oligonucleotides fixed on an electronically conductive polymer support. Consequently, the measurement of the fixation of the OM side chains on an electrode, under given experimental conditions, makes it possible to predict the quantity of oligonucleotides which will be fixed on another electrode (of the same matrix or of another matrix), under the same experimental conditions. The use of the POM conjugates and of the copolymers in accordance with the invention makes it possible to obtain electrodes constituting internal controls, and allowing a qualitative and / or quantitative control of the attachment of X molecules to a solid support constituted by the surface of an electrode carrying an electronic conductive polymer. This control of the attachment of the X molecules can be carried out at any time (after depositing them or during use). The molecules X whose attachment can be detected by using a copolymer in accordance with the invention can be of varied nature; advantageously, they comprise at least one oligonucleotide chain O and / or a molecule of interest M which may be identical to or different from those of the copolymer according to the invention used. A polymer according to the invention can be used to detect and / or quantify a molecule X which constitutes one of the side chains of the same polymer, or else of a polymer of the same formula, deposited on the same electrode, or else on another electrode of the same or a different matrix. It can also be used to detect and / or quantify a molecule X belonging to a different polymer, which can also be deposited on the same electrode, on another electrode of the same matrix or of a different matrix.

This detection and / or quantification can be carried out, for example, by detecting the oligonucleotide chains O of the copolymers in accordance with the invention by hybridization with a labeled probe, or else by using on one or more of the electrodes of the matrix, copolymers in accordance with l the invention comprising a molecule of interest M constituting a marker easy to detect (for example biotin, or a fluorescent marker).

The present invention will be better understood with the aid of the additional description which follows, which refers to examples of preparation and use of macromolecules and copolymers in accordance with the invention. EXAMPLE 1 SPECTRAL AND CHROMATOGRAPHIC PROPERTIES OF PEPTIDES

To illustrate the problems posed by the detection of a peptide of biological interest, a commercial synthetic peptide is used (ref. A2532 SIGMA-ALDRICH Chemistry), of molar mass 1652.1 Da, corresponding to fragment 1 1-24 of FACTH (adrenocortotrophic hormone). According to the manufacturer's specifications, the preparation contains 61% peptide.

5 μm LICHROSPHER® RP-18E HPLC column is injected

125-4 MERCK, DARMSAT, DE) 25 μl of a 2 mg / ml solution of said peptide. The elution (lml / min) is carried out by the mixture A + B, (A = 5% ACN (acetonitrile)

25 mM TEAAc (triethyl ammonium acetate); B = 50% ACN 25 mM TEAAc) using a gradient from 0% to 100% B in 35 minutes.

A peak is observed at a retention time Rt = 2 minutes and a fairly broad peak at Rt = 10 minutes. The fractions corresponding to these two peaks are collected, concentrated and analyzed.

The fraction FI (Rt = 2 minutes) shows an absoφtion At _{220 nm} = 1.033. On the other hand, at _{275 nm} is of the same order as the background noise (0.005). This fraction therefore contains salts (at ₂₂ o _nm ^non specific).

The fraction F2 (Rt = 10 minutes) has an absoφtion At _{220 nm} = 1.90 and At _{275 nm} = 0.073. This fraction therefore contains the peptide.

This illustrates the fact that UV detection around 215 at 220 nm does not, on its own, make it possible to differentiate on the chromatogram the signal due to the peptide from the parasitic signals of other substances.

The commercial synthetic peptide (ref. A 0673 SIGMA-ALDRICH CHEMISTRY), of molar mass 2465.7 Da, corresponding to the ACTH fragment (18-39), was analyzed in the same way by RP-HPLC. According to the manufacturer, the sample contains 82% peptide.

The HPLC analysis is carried out under the same conditions as above, and the fractions are detected in U.V. at 215 nm. A major peak is observed for Rt = 18.45 minutes; this peak is finer than that observed for the ACTH fragment (11-24).

This experiment shows that two different peptide fragments have peaks characterized by very different retention times. The comparison of the HPLC profiles also shows that the peaks, as in the case of ACTH (11-24), can be widened. EXAMPLE 2 SYNTHESIS OF AN OLIGONUCLEOTIDE DOUBLE MODIFIED BY A PYRROLE RESIDUE AND AN AMINOALKYL ARM.

A modified oligonucleotide of sequence: 5'HO-dC ^{pyITO, c} - (T) ₁ op-dC ^aminohcxyl -p-dTOH3 'is synthesized on a solid support (CPG (Controled pore Glass)) by the method called "phosphite-phosphoramidite" , described by BEAUCAGE and LYER, [Tetrahedron., 48, 2223-2311, (1992)].

The main stages of this synthesis are indicated below.

An aminoalkyl phosphoramidite derived from 5'-O- (4,4'-dimethoxytrityl) -N-4- (6-aminohexyl) -2'-deoxycytidine is prepared [ROGET et al. Nucleic Acids Res., 17, 7643-7650, (1989)].

This aminoalkylphosphoramidite is obtained in two stages: the primary amine of the alkylamine arm carried by the nucleoside is protected by a trifluoroacetyl group, followed by phosphitylation of the hydroxyl at 3 'by a process analogous to that described by SPROAT and al. [Nucleic Acids Res. 15, 6181-6196, (1987)], for the phosphoramidite of 5'-trifluoracetamido-2 ', 5'-dideoxythymidine by SPROAT et al. [Nucleic Acids Res. 15, 6181-6196, (1987)].

The aminoalkylphosphoramidites thus obtained are coupled on a column of a DNA synthesizer, via thymidines previously fixed on this column.

Other aminoalkyl phosphoramidites such as those described by AGRAWAL et al., [Nucleic Acids. Res., 14, 6227, (1986)]; CONNOLLY, [Nucleic Acids. Res. 15, 3131, (1987)]; BEAUCAGE and LYER, [Tetrahedron. 49, 1925-1963, (1993)] can also be used. The phosphoramidite of thymidine is then condensed on

T aminoalkyl-phosphoramidite attached to the column. This step is repeated until a 10-mer oligonucleotide chain is obtained.

Condensed at the 5 'end of the oligonucleotide thus obtained, a phosphoramidite of 5'-O- (4,4'-dimethoxytrityl) -N-4- (6-aminohexyl -) - 2'-deoxy- cytidine on which a pyrrole residue has been grafted, according to the protocol described in PCT application WO 94/22889 and by LIVACHE et al., [Nucleic Acids Res. 22, 2915-2921, (1994)].

The oligonucleotide is deprotected in concentrated ammonia

(16 hours at 55 ° C) and purified by HPLC on a column LiChrospher ^® RP-18 250-10 (10 .mu.m) (Merck, Darmstat, DE) with an acetonitrile gradient in triethylammonium acetate 50 mM ( buffer A: 5% acetonitrile, buffer B: 50% acetonitrile; flow rate 5 ml / minute, gradient from 10% B to 25% B in 20 minutes), according to the method described in: "Oligonucleotide synthesis: A practical approach. Ed MJ Gait. IRL Press, Oxford ”.

The fractions corresponding to a majority peak (retention time greater than 15 minutes) are evaporated. After evaporation, the oligonucleotide obtained is desalted by filtration on a NAP-5® column (PHARMACIA-LKB BIOTECHNOLOGY, UPPSALA, Sweden).

EXAMPLE 3 PREPARATION OF AN ACTIVE OLIGONUCLEOTIDE IN THE ESTER FORM OF N-HYDROXY SUCCINIMIDE. The modified oligonucleotide (hereinafter referred to as pyr-Tιo-NH2) prepared according to the method described in Example 2 above, is transformed into its activated intermediate (N-hydroxysuccinimide ester) corresponding according to the following protocol:

The lyophilized pyr-TjQ-NH2 oligonucleotide (10 to 25 nmol) is taken up in 12 μl of 50 mM N- (3-sulfopropyl) moφholine (MOPS) buffer pH 7.0. To this solution are added 28 μl of dimethylformamide containing 4 μmol of dissuccinimidyl suberate (DSS PIERCE ROCKFORD, IL), and the mixture is left under mechanical stirring (16 hours at 4 ° C or 5 hours at 20 ° C). The reaction mixture is deposited on an NAP-5® column equilibrated with water, and the column is eluted with water according to the protocol recommended by the manufacturer. The excluded fraction (1 ml) is extracted 5 times with 1 ml of n-butanol. On each extraction, it is centrifuged, the upper phase (organic) is eliminated and the lower phase (aqueous) is kept. At the last extraction, the N-hydroxy succinimide ester (hereinafter referred to as pyr-Tio-NHS) precipitated at the bottom of the tube is dried under vacuum (SPEED-VAC) and then stored at -20 ° C (preferably less 24 hours) until use.

Analytical HPLC of an aliquot of the reaction mixture at different reaction times is carried out on a LICHROSPHER® RP-18E / 125-4 column (5 μm) (MERCK, DARMSTAT, DE); elution is carried out with a gradient of acetonitrile in 50 mM triethylammonium acetate (buffer A: 5% acetonitrile, buffer B: 50% acetonitrile); flow rate 1 ml / min, gradient from 10% B to 30% B in

20 minutes, then from 30% B to 50% B in 15 minutes. This chromatographic analysis shows the disappearance of the peak of the pyr-Ti Q-NH ₂ oligonucleotide (retention time approximately 19 minutes) and the appearance of a peak (retention time approximately 27 minutes) corresponding to the ester of N-hydroxy succinimide (called pyr-Tio-NHS). EXAMPLE 4: COUPLING OF A MODIFIED OLIGONUCLEOTIDE ON PEPTIDES

A) Coupling on the ACTHCL1-24 fragment) t

20 nmol of pyr-Tio-NHS obtained as described in Example 3 are taken up in 50 ml of MOPS buffer (50 mM, pH 7.8). We add the peptide

ACTH (11-24) (SIGMA, St Louis, United States) (26 nmol, 1.3 eq.) In 50 μl of buffer

MOPS pH 7.8. Incubate overnight at 4 ° C.

The disappearance of pyr-Tκ) -NHS is observed in analytical HPLC under conditions identical to those of Example 3, and the appearance of a majority peak corresponding to the oligonucleotide-peptide conjugate (retention time approximately 26, 4 minutes.

The reaction mixture is purified by semi-preparative HPLC on a LICHROSPHER® RP-18E / 125-4 column (5 μm), by a gradient of acetonitrile in 50 mM triethylammonium acetate. The elution is carried out under the same conditions as the analytical HPLC described in Example 3.

The oligonucleotide-peptide conjugate is detected by UV measurement at 265 nm and is collected in the fraction corresponding to a retention time of between 24 and 25.5 minutes; this fraction is dried by evaporation (SPEED-VAC). This gives about 3.7 nmol of conjugate called pyr-TiQ-ACTH (1-24).

B) Coupling on the ACTH fragment (18-39);

The pyr-Tio-NHS oligonucleotide-ester (EX. 3) (approximately 20 nmol) is coupled with approximately 35 nmol, ie approximately 1.8 eq. of the ACTH fragment (18-39) (SIGMA, St Louis, United States), and the coupling product, called pyr-Ti Q-ACTH (18-39), is purified according to the protocols described in A) above. above.

The pyr-Tιo-ACTΗ conjugate (18-39) is detected in the fraction corresponding to a retention time between 26 to 27 minutes. After drying this fraction, about 7 nmol of the conjugate is obtained.

EXAMPLE 5 SYNTHESIS OF A PYRROLE-T ₁₀ -BIOTIN CONJUGATE: A modified oligonucleotide of pyr-Ti _Q -NH2 sequence (10 to 20 μmol) is treated with an excess of dissolved biotin-NHS (approximately 50 equivalents) (SIGMA) in 20 μl of dimethylformamide in a carbonate buffer (1 M) at pH 9; incubated for 30 minutes at 20 ° C.

The reaction mixture is purified on an exclusion column (NAP PHARMACIA) and the pyr-Tιθ-bi ° conjugate (Rt = 23 minutes) is separated from the residual pyr-Tιo-NH2 oligonucleotide (Rt = 18 minutes) by analytical HPLC under the conditions of Example 3. The fractions are detected at 265 nm.

EXAMPLE 6: ELECTROPOLYMERIZATION OF PYR-T CONJUGATES ₁₀ -

PEPTIDE, AND PYR-T ₁₀ -BIOTIN. The conjugates synthesized as described in Examples 4 A), 4

B) and 5, are deposited, by electropolymerization on silicon micro-electrodes coated with gold.

These micro-electrodes are arranged so as to form a matrix in the form of a "checkerboard"; this matrix is formed by square micro-electrodes of 50 μm side arranged in 5 columns and 4 lines according to the following diagram (n = 5 columns and p = 4 lines). The micro-electrodes are identified by their coordinates

(i; j) -

The electropolymerization is carried out by immersing the electrode matrix in a medium containing the conjugated pyrrole-oligonucleotide-biomolecule concerned, and pyrrole (molar ratio Pyrrole / conjugate = around 10,000), in a LiClO ₄ solution 0.1 M. One connects the electrode on which one wishes to carry out the deposit to a potentiostat, and one carries out cycles between -0.35 V and +0.85 V (potentials measured compared to a calomel electrode connected to the cell d electrolysis and connected to the potentiostat) at a speed of 100 mV / s. The counter electrode consists of a platinum wire.

Some of the micro-electrodes are covered with a copolymer formed from polypyrrole and a conjugate:

- pyr-T ₁₀ -ACTH (18-39): electrodes (1; 4) and (3; 4)

- pyr-Ti Q-bio: electrodes (2; 3) and (4; 4). The remaining electrodes are either left in their initial state, that is to say that the gold deposit remains unchanged (OR), or else covered with unmodified polypyrrole (PP); these electrodes constitute negative controls making it possible to evaluate the specificity of the reactions carried out on the matrix.

Table I below shows schematically the microelectrode array, and the location of the various deposits. TABLE I

OR: no deposit

PP: Polypyrrole deposit EXAMPLE 7: HIGHLIGHTING OF ELECTROPOLYMERIZED BIOMOLECULES ON THE ELECTRODES

The electrode matrix produced in Example 6 is incubated in the presence of a streptavidin-phycoerythrin conjugate (commercial streptavidin-R-phycoerythrin solution MOLECULAR PROBES, at 1 mg / ml diluted 1/20 in 10 mM phosphate buffer pH 7, 4 containing 0.5M NaCl and 0.05% TWEEN 20).

Observation under an epifluorescence microscope reveals that the fluorescence is localized on the electrodes (2; 3) and (4; 4). There is no parasitic fluorescence on the other electrodes, which shows that the pyrrole-oligonucleotide-biotin conjugate has specifically fixed on the desired target electrodes.

The same microelectrode array is incubated in the presence of a biotinylated antico spécifiques specific for the C-terminal part of ACTH peptide 18-39 (anticoφs ACR-17-bio: CIS BIO INTERNATIONAL) then in the presence of a streptavidin / phycoerythrin conjugate to reveal the reaction product.

Observation under an epifluorescence microscope reveals that only the micro-electrodes carrying the pyrrole-oligonucleotide-ACTH conjugate, and those carrying biotin are fluorescent, which shows that the pyrrole-oligonucleotide-ACTH conjugate is specifically fixed on the electrodes target, that it is accessible to an anticoφs, and that its antigenic properties are preserved. EXAMPLE 8:

Using the method described in Example 4, the ACTH fragment (18-39) is coupled to an oligonucleotide-pyrrole ester of N- hydroxysuccimmide, prepared by the method described in Example 3, from an oligonucleotide-pyrrole of sequence 5'pyr-T9-NH2, synthesized according to the protocol described in Example 2.

There are thus available POM type conjugates: I. pyr-T ₁₀ -ACTH (l l-24)

IL pyr-T ₁₀ -ACTH (18-39)

III. pyr-T ₉ -ACTH (18-39)

IV. pyr-TiQ-Biotin

V. pyr-T ₅ -HCVG (formed from the association of a T ₅ arm and a specific sequence of the genome of the HCV virus).

By following the electropolymerization process of Example 6, an electrochemical deposition of the conjugates I to V is carried out on a network of square micro¬ electrodes with a side of 100 μm.

Table II below shows schematically the microelectrode array, and the location of the various deposits.

Some electrodes are left uncovered (OR) or covered with unmodified polypyrrole (PP).

This microelectrode plate is incubated with an oligonucleotide probe of sequence dC ^Blotme (dA) 9 prepared and purified according to Example 2, and using the phosphoramidite biotin described by ROGET et al. [Nucleic Acids Res., 17, 7643-7650 (1989)].

Hybridization is carried out with approximately 8 pmol of oligonucleotide probe in 200 μl of hybridization buffer (0.1 M phosphate buffer, pH 7.4 containing 0.5 M Nacl / TWEEN, 30 min at 20 ° C then 30 min at 4 ° C.

It is then revealed by incubation (10 min at 4 ° C.) in a streptavidin-phycoerythrin solution (commercial streptavidin-R-phycoerythrin solution MOLECULAR PROBES at 1 mg / ml) diluted 1/20 in hybridization buffer.

After examination under a fluorescence microscope, it is observed that the electrodes (1; 2) and (2; 5) are strongly positive, that the electrodes (1; 3), (1; 4), (2; 3) and (2 ; 4) are positive, while the electrodes (1; 5) and (2; 2) show fluorescence weaker ; all the other electrodes and, among others (1; 7) and (2; 7) do not exhibit fluorescence above the background noise.

The conjugate IV (pyr-T] o-Biotin) serves as a positive revelation control. When using the plate for the first time, it certifies revelation with the streptavidin / phycoerythrin complex. The 4 microelectrodes comprising the P-O-M type conjugate in which O is an oligonucleotide of sequence T \ Q. (1; 3), (1; 4) (2; 3) and (2; 4) exhibit positive hybridization, which makes it possible to certify that the compound of P-O-M type has been copolymerized on these electrodes. The weak hybridization observed on the electrodes (1; 5) and (2; 2) and the absence of hybridization on the electrodes (1; 7) and (2; 7) shows the selectivity of the hybridization and illustrates the limit. lower for the size of the oligonucleotide part of the conjugate allowing this dilution by hybridization, which can be estimated between 5 and 9 nucleotides in length. EXAMPLE 9:

The POM Pyr-T] Q-Bio type conjugate described in Example 5 is used, in solution in water at a concentration of 140 AU ₂₆₅ / ml, which makes it possible to determine the molar concentration, ie 1.17 μmole / ml (with ε ₂₆₅ = 120,000) thanks to the UV absorption properties of the O part of this conjugate.

A dilution range of the conjugate is carried out sequentially (1/5; 1/25; 1/50; 1/250; 1/1250) in water and 10 μl of each dilution are added to 300 μl of pyrrole solution 20 mM in 0.1M LiClO ₄ .

Using solutions containing varying concentrations of Pyr-Ti Q-Bio conjugate, copolymerizations are carried out on microelectrodes as described in Example 6. The pyrrole / conjugate molar ratio is different for each electrode.

Table III below diagrams the microelectrode array, and the location of the various deposits. TABLE III

The revelation is carried out by incubation in a 1/20 ^{th solution} of streptavidin / phycoerythrin in a 10mM phosphate buffer pH 7.4 containing 0.5M NaCl and 0.05% Tween 20. The electrode plate is observed under a epifluorescence microscope coupled to a CCD camera (HAMAMATSU), itself connected to a microcomputer provided with an image analysis program.

The exposure time can be set to vary the sensitivity of the detection. Using exposure times of 0.5 s, 1 s, 2 s, 4 s, 8 s, 16 s, we observe that at least the three contiguous pads (1; 2), (1; 3) ( 1; 4) always give fluorescence signals, which are of increasing intensity (1; 2> (1; 3)> 1; 4). The studs (1; 1) and (2; 1) make it possible to appreciate the background noise).

The signals provided by the pads (1; 2) and (2; 2), (1; 3) and (2; 3) etc. are of the same order two by two, which makes it possible to assess the reproducibility.

This makes it possible to show that the fluorescence intensities are correlated with the initial quantities of POM type compound (originally quantified by absoφtion at 265 nm) introduced into the different electrolyte solutions. EXAMPLE 10 a) Variable quantities of a pyrrole-oligonucleotide conjugate, (pyrrole-Tjo-k-ras), of formula Pyr-5 '(Tι o) -k-ras (28-41> in which (28- 41) represents the sequence of nucleotides 28 to 41 of the sense strand of the human k-ras sequence carrying a G- »A mutation on nucleotide 34, were electropolymerized, as described in example 6, in the presence of a quantity pyrrole constant (600 μl of 20mM pyrrole in LiClO ₄ , i.e. 1.2 x 10 ^"5 mol), on 5 different microelectrodes of a plate of square microelectrodes (50 μm x 50 μm) coated with gold, as indicated on Table IV below. TABLE IV

b) On 5 other microelectrodes of the same wafer, the procedure is the same with the POM pyrrole-Tiobiotin conjugate, using the range illustrated in table V below, and in FIG. 1 (b): TABLE V

The plate is incubated for 30 min at 45 ° C., in the presence of the oligonucleotide complementary to the sequence k-ras (28-41), biotinylated at 5 '. A 2UA ₂₆₀ / ml solution of biotinylated oligonucleotide is used, diluted to 1 / 1000th in 10 mM PBS buffer pH 7.4 containing 0.5M NaCl and 10 mM EDTA. After washing (20 ° C) with 10 mM PBS buffer containing 0.5M NaCl and 0.05% TWEEN

20, the plate is incubated (10 min at 20 ° C.) in a solution of streptavidin-phycoerythrin (commercial solution streptavidin-R-phycoerythrin MOLECULAR

PROBES 1 mg / ml) diluted 1/20 in 10 m PBS buffer, pH 7.4 containing

0.5M NaCl and 0.05% TWEEN 20.

The plate is observed under an epifluorescence microscope

(OLYMPUS) equipped with a CCD camera (integration time t _s = 2s; gain = x 4) connected to a computer equipped with image analysis software (IMAGE PRO).

The fluorescence measurements of the pyrrole-Ti Q-k-ras and pyrrole-Tiobiotin ranges are illustrated in FIG. 1:

(- D - = k-ras; - • - = biotin), which represents the fluorescence on each electrode as a function of the initial quantity of conjugate present in the cell during the electropolymerization, and therefore of the molar ratio pyrrole / conjugate. This experiment shows that the choice of the pyrrole / P-OM or pyrrole / oligonucleotide molar ratio makes it possible to control the quantity of molecules grafted onto each electrode. EXAMPLE 11:

On a matrix of micro-electrodes formed by 50 μm square electrodes (see example 6), a pyr-Ti Q-Biotin conjugate is deposited by electro-polymerization according to the protocol of example 6, and according to the arrangement (shown by the coordinates i / j) indicated on the first 2 lines of table VI below. 2 series of identical deposits, B _n and B _n 'are made: deposits B, and B,' are made successively using two solutions prepared from the same conjugate solution; a rinse of the electrode and of the cell is inserted between each deposit.

The same is done with B ₂ and B ₂ \ etc. Each series of deposits is made using dilutions (in water) from 1/5 to 1/5, such as [B ₂ ] = [B ,] / 5; [B ₃ ] = [B ₂ ] / 5. These dilutions are obtained from a stock solution of Pyr-Ti Q-Biotin estimated at 125 mmol / 1 (measurement of OD ₂₆₅ by taking ε ₂₆₅ = 120,000). 8 μl of each dilution is used in 600 μl of electro-polymerization solution (20 mM pyrrole in 0.1 M LiC10 ₄ ). The concentrations of Pyr-Ti-biotin conjugate (C), the [Pyrrole] / [Pyr-Ti-biotin] (R) ratios, and the quantities of Pyr-Tj () - biotin conjugate initially present in the electropolymerization cell , for each group Bi / Bi '; B ₂ / B ₂ '; etc. are shown in Table VI below.

After washing the electrode plate, by incubation discloses the presence of a streptavidin-phycoerythrin conjugate (10 min. At 20 ° C, diluted commercial solution to l / ^10th in lOmM PBS buffer pH 7.4, containing 0.5M NaCl and 0.05% TWEEN 20).

After rinsing with the same buffer, the plate is observed under a fluorescence microscope, equipped with a HAMAMATSU CCD camera and a IMAGE PRO image analysis software (integration time tj = 2 s; gain x 4). Using this software, the fluorescence of each deposit can be measured point by point.

The results are illustrated by Table VII below, where the fluorescence for each deposit is indicated, in arbitrary units of fluorescence (UFA) according to the coordinate system (i; j) of Table VI, and by Figure 2, which represents the fluorescence in UFA as a function of the initial quantity of pyr-biotin conjugate in the electropolymerization cell (D = series B; • = series B ').

TABLE VII

These results clearly show a relationship of direct proportionality between the response (fluorescence) and the initial quantity of pyrrole-Tiobiotin conjugate (or the initial ratio [pyrrole] / [pyr-Ti () - biotin], the pyrrole concentration being constant) in the electropolymerization mixture.

The use of compound of the P-O-M type therefore makes it possible to easily control the amount of pyrrole conjugate (pyr-TiQ-bio) present in an electrolyte solution, and to carry out ranges of calibration of electrode arrays.

Claims

1) Macromolecule of formula (I):

P-O-M (i) in which:

M represents a molecule of interest;

O represents an oligonucleotide chain;

P represents a monomer of an electronic conductive polymer.

2) Macromolecule according to claim 1, characterized in that the monomer P represents a pyrrole group.

3) Macromolecule according to any one of claims 1 or 2, characterized in that the oligonucleotide chain O consists of a single-stranded oligonucleotide.

4) Macromolecule according to any one of claims 1 or 2, characterized in that the oligonucleotide chain O consists of a double-stranded oligonucleotide over at least part of its length.

5) Macromolecule according to any one of claims 1 to 4, characterized in that the oligonucleotide chain O comprises at least 6 nucleotides, and in that its percentage in (G + C) is less than or equal to 70%. 6) Process for preparing a POM macromolecule according to any one of claims 1 to 4, from a mixture comprising said macromolecule as well as the reagents PO and M from which it was formed, which process is characterized in that '' It comprises at least one step during which one proceeds to the fractionation of said mixture, by all means making it possible to separate the fractions comprising M and POM respectively from the fraction comprising PO and a step during which one proceeds to detecting the fraction comprising POM, and / or determining the amount of POM by detecting and / or measuring a parameter associated with the oligonucleotide O and not associated with the molecule of interest M. 7) Method according to claim 6, characterized in that said parameter associated with the oligonucleotide O and not associated with the molecule of interest M is the absorption in ultraviolet, at a wavelength comprised e between 240 and 270 nm.

8) Copolymer, characterized in that at least one of its units consists of a macromolecule according to any one of claims 1 to 5. 9) An electrode characterized in that its surface carries at least one copolymer according to claim 8.

10) Array of electrodes, characterized in that it comprises at least one electrode according to claim 9. 11) Array of electrodes according to claim 10, characterized in that it comprises at least two electrodes differing from each other by at at least one of the monomers P, of the oligonucleotides O and / or of the molecules M present on their surface.

12) Array of electrodes according to claim 10, characterized in that it comprises at least two electrodes differing from each other by the amount of oligonucleotides O and / or of molecules M per unit area.

13) An electrode matrix according to any one of claims 10 to 12, characterized in that it further comprises at least one electrode covered with a PCE / oligonucleotide copolymer.

14) Use of at least one copolymer according to claim 8 for the qualitative and / or quantitative control of the fixing of at least one molecule X on an electrode.

15) Use according to claim 14, characterized in that said molecule X comprises at least one of the constituents P, O, or M of a macromolecule according to claim 1. 16) Use according to any one of claims 14 or 15, characterized in that the copolymer according to claim 8, and the molecule X are on the same electrode.

17) Use according to any one of claims 14 or 15, characterized in that the copolymer according to claim 8, and the molecule X are on two different electrodes.

18) Use according to claim 17, characterized in that the 2 electrodes belong to 2 different matrices.